Prostate cancer--the most commonly diagnosed cancer in men worldwide--can have a substantial effect on quality of life, regardless of the route the cancer takes. The serum PSA assay is the current ...gold standard option for diagnosing prostate cancer. However, a growing body of evidence suggests that PSA screening for prostate cancer results in extensive overdiagnosis and overtreatment. It is increasingly evident that the potential harm from overdiagnosis (in terms of unnecessary biopsies) must be weighed against the benefit derived from the early detection and treatment of potentially fatal prostate cancers. Rapid screening methods have been used to analyse glycosylation patterns on glycoproteins in large cohorts of patients, enabling the identification of a new generation of disease biomarkers. Changes to the expression status of certain glycan structures are now widely thought to be common features of tumour progression. In light of this development, much research has focused on the potential role of altered PSA glycosylation patterns in discriminating between significant and insignificant prostate cancers, with the aim of developing a more reliable diagnostic tool than the current serum PSA test.
The diagnosis and treatment of prostate cancer (PCa) is a major health-care concern worldwide. This cancer can manifest itself in many distinct forms and the transition from clinically indolent PCa ...to the more invasive aggressive form remains poorly understood. It is now universally accepted that glycan expression patterns change with the cellular modifications that accompany the onset of tumorigenesis. The aim of this study was to investigate if differential glycosylation patterns could distinguish between indolent, significant, and aggressive PCa. Whole serum
-glycan profiling was carried out on 117 prostate cancer patients' serum using our automated, high-throughput analysis platform for glycan-profiling which utilizes ultra-performance liquid chromatography (UPLC) to obtain high resolution separation of
-linked glycans released from the serum glycoproteins. We observed increases in hybrid, oligomannose, and biantennary digalactosylated monosialylated glycans (M5A1G1S1, M8, and A2G2S1), bisecting glycans (A2B, A2(6)BG1) and monoantennary glycans (A1), and decreases in triantennary trigalactosylated trisialylated glycans with and without core fucose (A3G3S3 and FA3G3S3) with PCa progression from indolent through significant and aggressive disease. These changes give us an insight into the disease pathogenesis and identify potential biomarkers for monitoring the PCa progression, however these need further confirmation studies.
Classifying indolent prostate cancer represents a significant clinical challenge. We investigated whether integrating data from different omic platforms could identify a biomarker panel with improved ...performance compared to individual platforms alone. DNA methylation, transcripts, protein and glycosylation biomarkers were assessed in a single cohort of patients treated by radical prostatectomy. Novel multiblock statistical data integration approaches were used to deal with missing data and modelled via stepwise multinomial logistic regression, or LASSO. After applying leave‐one‐out cross‐validation to each model, the probabilistic predictions of disease type for each individual panel were aggregated to improve prediction accuracy using all available information for a given patient. Through assessment of three performance parameters of area under the curve (AUC) values, calibration and decision curve analysis, the study identified an integrated biomarker panel which predicts disease type with a high level of accuracy, with Multi AUC value of 0.91 (0.89, 0.94) and Ordinal C‐Index (ORC) value of 0.94 (0.91, 0.96), which was significantly improved compared to the values for the clinical panel alone of 0.67 (0.62, 0.72) Multi AUC and 0.72 (0.67, 0.78) ORC. Biomarker integration across different omic platforms significantly improves prediction accuracy. We provide a novel multiplatform approach for the analysis, determination and performance assessment of novel panels which can be applied to other diseases. With further refinement and validation, this panel could form a tool to help inform appropriate treatment strategies impacting on patient outcome in early stage prostate cancer.
In this study, we built a novel statistical model across multiple omic platforms to predict indolent and aggressive prostate cancer. We demonstrate using ROC, calibration and decision curves that our combined biomarker panel significantly improves on the prediction of indolent disease compared to current clinical features. This will inform appropriate treatment strategies impacting on patient outcomes in early stage prostate cancer.
Recent exploitation of the avian immune system has highlighted its suitability for the generation of high-quality, high-affinity antibodies to a wide range of antigens for a number of therapeutic and ...biotechnological applications. The glycosylation profile of potential immunoglobulin therapeutics is species specific and is heavily influenced by the cell-line/culture conditions used for production. Hence, knowledge of the carbohydrate moieties present on immunoglobulins is essential as certain glycan structures can adversely impact their physicochemical and biological properties. This study describes the detailed N-glycan profile of IgY polyclonal antibodies from the serum of leghorn chickens using a fully quantitative high-throughput N-glycan analysis approach, based on ultra-performance liquid chromatography (UPLC) separation of released glycans. Structural assignments revealed serum IgY to contain complex bi-, tri- and tetra-antennary glycans with or without core fucose and bisects, hybrid and high mannose glycans. High sialic acid content was also observed, with the presence of rare sialic acid structures, likely polysialic acids. It is concluded that IgY is heavily decorated with complex glycans; however, no known non-human or immunogenic glycans were identified. Thus, IgY is a potentially promising candidate for immunoglobulin-based therapies for the treatment of various infectious diseases.
Reconciling the Structural Attributes of Avian Antibodies Conroy, Paul J.; Law, Ruby H.P.; Gilgunn, Sarah ...
Journal of biological chemistry/The Journal of biological chemistry,
05/2014, Letnik:
289, Številka:
22
Journal Article
Recenzirano
Odprti dostop
Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the ...diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.
Antibodies from alternative immune hosts provide insights into novel mechanisms of antibody diversity in restricted germ-line repertoires.
The high-resolution crystal structures of the first two chicken single chain antibodies (scFv) with prototypical binding sites are described.
Chickens exhibit unique canonical classes in the CDRL1.
Aves employ distinct mechanisms to generate diversity resulting in unique binding-site topologies.
•Upstream process parameters leading to HCPs in bioprocessing of mAbs in CHO cell lines.•Removal of HCPs in downstream bioprocessing.•Current methodologies for tracking and characterisation of CHO ...HCPs.•Identification of ‘problematic’ HCPs.
Monoclonal antibodies hold a steadfast lead in the ever-expanding biologics marketplace and have revolutionized the treatment of a wide variety of illnesses. The prominence of mAbs as therapeutic agents brought with it the need for large scale production of these drugs, which in turn highlighted the need for improvements in cell culture processes to raise product titres. Increased product titres shifted bioprocessing concerns downstream as with increased titre brought along the increased expression of unwanted host cell proteins (HCPs). HCPs are a highly diverse range of proteins. While some HCPs can be degradative to the product itself, others could induce an unwanted immune response compromising the safety and efficacy of the biologic. Enzyme-linked immunosorbent assays (ELISAs) are currently the gold standard for release testing for HCPs. ELISAs provide quantitative measurement of total HCP levels but have several limitations. Industry has shifted towards the use of orthogonal methods to support process development and validation with a particular focus on analytical tools such as LC–MS/MS.
This review discusses the current methods for identification and analysis of problematic HCPs in CHO cell lines used for mAb bioprocessing.
Highly sensitive and label free detection of prostate specific antigen (PSA) still remains a challenge in prostate cancer diagnosis. In this paper, we propose a sensitive electrochemical immunosensor ...based on electrocatalytic platinum nanoparticles conjugated to a recombinant scFv antibody. Gold disc electrodes functionalised with a l-Cysteine (Cys) self-assembled monolayer (SAM) were used to covalently bind PSA specific monoclonal antibody (anti-PSA) using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. Immunosensing was completed using sandwich-type immunoreaction of the PSA–antigen (1–30ng/mL) between anti-PSA immobilized on the l-Cys modified electrode using label free electrochemical impedance (EIS) technique. Furthermore, highly specific in-house generated scFv fragments as receptor proteins were utilised for one step site-directed immobilisation on the surface of platinum nanoparticles (PtNPs). To improve the sensitivity of the immunoassay, these scFV labelled electrocatalytic PtNPs were then used for covalent hybridisation to the PSA modified electrode and then applied in a hybridisation assay to determine the concentration of the PSA by measuring the faradaic current associated with reduction of peroxide in solution. Semi-log plots of the PSA concentration vs. faradaic current are linear from 1 to 30ng/mL and pM concentrations can be detected without the need for molecular, e.g., PCR or NASBA, amplification.
•A sensitive electrochemical immunosensor based on based on capturing electrocatalytic platinum nanoparticles in a sandwich assay.•The in-house generated scfV fragments combine the high affinity and specificity of full length antibodies.•Binding of the platinum nanoparticles labelled with the recombinant scFv antibodies changed the impedance (both resistance and capacitance) properties of the interface.
Abstract Clotting factor replacement therapy has proven a highly effective means of treating haemophilia A and B. But treatment involves frequent and lifelong infusion of factor concentrates and is ...generally prophylactic rather than curative. It is also extremely expensive, associated with inhibitor formation and does not fully abolish the potential for spontaneous bleeding. Gene therapy offers a potential cure for haemophilia, with the possible continuous expression of a clotting factor gene following the administration of a viral vector carrying the appropriate gene. Recent clinical trials of gene therapy for haemophilia have proven positive in selected patients and new studies are underway.
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Multi-analyte, multi-step LoaD depicting the spatial distribution of various unit operations, liquid chambers and valves.
•Multi-step sample-to-answer process automation using purely ...rotational flow control.•Dissolvable film valve release-frequency definition enhanced more than 3 fold over state-of-the-art.•Novel valve architecture for significantly higher integration density of operations.•Triplex on-disc detection of Prostate cancer multi-biomarker panel using ELISA with controls.•Flexibility of design for operation of variants of the ELISA format.
Early diagnosis and prognosis of prostate cancer (PCa) is increasingly moving towards evaluations based on a strategic combination of biomarkers. The ability to parallelize multi-parameter detection on the same device is hence considered as a substantial improvement compared to conventional, single-marker methods such as total prostate-specific antigen (t-PSA). We demonstrate full rotational control of a centrifugal microfluidic system which is geared to automate multi-step/multi-reagent protocols underlying PCa detection based on a multi-marker panel on a widely autonomous, cost-efficient and rugged point-of-care instrument. Such comprehensive fluidic integration is enabled by a novel type of dissolvable-film (DF) valve which significantly enhances the definition of its critical release frequency by a factor of more than 3 (average spread of ±1.8Hz), and thus substantially enhances process reliability and integration density on our “Lab-on-a-Disc” (LoaD) platform. This siphon-based centrifugo-pneumatic flow control scheme can be attuned merely by temporal modulation of the spin rate to parallelize three assay protocols in a sample-to-answer fashion; in addition, we demonstrate flexibility of application without redesigning of the cartridge towards variants of the common Enzyme-Linked Immuno-Sorbent Assay (ELISA) format. As a pilot application, we further implement liquid handling automation for the detection of two distinct PCa markers, free PSA and human epidermal growth factor receptor-type 2, (HER2), using two types of ELISA i.e. sandwich and competitive, respectively.
The proportion of prostate-specific antigen (PSA) circulating in the free form can help to differentiate prostate cancer from benign prostate disease. In this paper, novel highly specific ...single-chain antibody (scAb) fragments were conjugated to magnetic particles (MPs) for the recognition and magnetic separation of free PSA (fPSA). A sandwich-type immunoassay method was developed, in which fPSA was captured by the scAb-conjugated MPs and labelled by a secondary antibody, conjugated to horseradish peroxidase (HRP) enzyme. The biosensor was constructed on carbon screen-printed electrodes (SPEs) modified with magnetic particles for increased surface area and fast reaction kinetics, thus enhancing binding capacity. The amperometric response was measured at −0.15 V, using hydrogen peroxide as the HRP substrate and hydroquinone (HQ) as the electrochemical mediator. The magneto-immunosensor demonstrated a sensitivity of 0.5 ng mL
−1
, with a linear detection range up to 10 ng mL
−1
of fPSA.
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